The present invention relates to a bioactive extract containing a natural non-polar and non-proteinaceous fluorescent dye extracted from alcoholic extract of ovarian tissue of a marine invertebrate Holothuria scabra. The invention also provides a process for the extraction, partial purification and characterization of this new non-polar dye. The marine invertebrate mentioned is especially the sea cucumber. Sea cucumbers are echinoderms, members of the group of spiny skinned animals that also includes starfishes and sea urchins. Scientifically called Holothurians, they have elongated tubular bodies which are rubbery and without bony skeletons. They have the following taxonomic position.
Sea cucumbers have following taxonomic position.
Amongst these orders sea cucumber Holothuria scabra belongs to:
Echinoderms are coelomate invertebrates which are exclusively marine, never colonial, are unsegmented with a basic pentameric radial, symmetry in the adult form, no head or brain, and distinguished from all other animals by structural peculiarities of skeleton and coelom. Class Holothuroidea has animals with body bilaterally symmetrical, usually elongated in the oral-aboral axis having mouth at or near one end and anus at or near the other end. The body surface is coarse, endoskeleton reduced to microscopic spicules or plates embedded in the body wall, mouth surrounded by a set of tentacles attached to water vascular system; podia or tube feet are usually present and locomotory; alimentary canal is long and coiled and cloaca usually with respiratory trees; sexes are usually separate and gonad single or paired tuft of tubules. They are sedentary forms either attached to hard substrate or burrow into soft sediments with anterior and posterior ends projected. There are more than 1000 species of holothuroids. They vary from 2 cm to 2 meter in length. They are among a few of the animals whose habitat is not restricted by ocean depths. Some of the species are reported to be making 50% of life forms at 4000 m and 90% at 8000-m depths. The species Holothuria scabra also called by some as Metriatyla scabra Jaegea is widely distributed in East Africa, Red Sea, Bay of Bengal, East India, Australia, Japan, South Pacific, Philippines, Indian Ocean and other Indo-Pacific regions. It is used for human/animal consumption in Sabah, Malaysia and Indonesia and other Indo-Pacific countries.
Fluorescent dyes are of immense value in the fluorescent probe industries to enable researchers to detect particular components of complex biomolecular assemblies, including live cells with exquisite sensitivity and selectivity (Iain D. Johnson. xe2x80x9cIntroduction to fluorescence techniquesxe2x80x9d Chapter 1.pp1 in The Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the United States of America, 1996). Fluorescent dyes are widely used in labeling of molecular probes for localizing biological structures by fluorescence microscopy e.g in immunoassays, labeling nucleotides and oligonucleotides for in situ hybridization studies, binding to polymeric microspheres and staining of cells for use in imaging studies. Dyes are also used for selective destruction of cells such as in the technique of photodynamic therapy. (Haughland, R. P and Kang, H. C. U.S. Pat. No. 4,774,339 of Sep. 27, 1988; Haughland, R. P and Kang, H. C. U.S. Pat. No. 5,248,782, Sep. 28, 1993).
Non-polar dyes are useful in detection of neutral lipids and other non-polar liquids. The fluorophores with the non-polar quality are most suitable for making conjugants with variety of biomolecules as per the requirement of the reseachers. Two non-polar probes namely NBD probes, NBD dihexadecylamine (D-69) and NBD hexadecylamine (H-429) are primarily been used for fluorescence detection of lipids separated by TLC (The Molecular probes Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the United States of America, 1996 pp.321-322). The same authors reported three non-polar derivatives of the bodipy fluorophore that offers an unusual combination of non-polar structure and long-wavelength absorption and fluorescence. These dyes are described to have potential applications as stains for neutral lipids and as tracers for oil and other non-polar liquids. For example BODIPY 493/503 (D-3922) is more specific for cellular lipid droplets then staining with Nile red (N-1142). BODIPY 505/515 (D-3921) rapidly permeates cell membranes of live Zebrafish embryos, selectively staining cytoplasmic yolk platelets (Mark Cooper, University of Washington quoted by Molecular probes The Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the United States of America, 1996 pp.322). Bodipy dyes have a small fluorescence, Stokes shift extinction coeffecients that are typically greater than 80,000 cmxe2x88x921 Mxe2x88x921 and high fluorescence quantum yields (0.94 for D-3921 in methanol) that are not diminished in water. Their photostability is generally high These Bodipy dyes are used in flash lamp-pumped laser dyes. Bodipy 665/676 (B-3932) can be excited using the 633 nm spectral line of the he-Ne laser or the 647 nm spectral line of the Ar-Kr laser for applications requiring long-wavelength fluorescence detection. (Richard P. Haughland, Molecular probes. The Handbook of Fluorescent probes and Research Chemicals 6th edition Printed in the United States of America, 1996 pp.321-322). Non-polar fluorescent dyes as dye component in industrial applications are reported in US patents (Fischer, Wolfgang; Deckers, Andreas; Guntherberg, Norbert; Jahns, Ekkehard; Haremza, Sylke; Ostertag, Werner; Schmidt, Helmut. U.S. Pat. No. 5,710,197 released on Jan. 20, 1998 entitled xe2x80x9cCrosslinked polymer particles containing a fluorescent dyexe2x80x9d; U.S. Pat. No. 5,304,493 dated Apr. 19, 1994 by Nowak, Anthony V Title xe2x80x9cMethod for detecting a marker dye in aged petroleum distillate fuels; U.S. Pat. No. 4,063,878 dated Dec. 20, 1977 by Weeks, Bruce W entitled xe2x80x9cApplying sublimation indicia to pressure sensitive adhesive tapexe2x80x9d).
At the website, http://www.uniulm.de/uni/fak/natwis/oc2/ak_wuert/publications.htm there are reported 39 publications of A. K. Wuerthner, most of which pertains to synthesis and design of chromatphores for refractive index materials and photorefractive organic glasses. About nine international patents are also disclosed (S. Beckmann, F. Effenberger, F. Wxc3xcrthner, F. Steybe, DE 44 16 476 (Nov. 16, 1995), WO 95/30679, Preparation of oligothiophenes for use in nonlinear optical materials; K-H. Etzbach, C. Krxc3xa4h, R. Sens, F. Wxc3xcrthner, DE 19.611.351 (Mar. 22, 1996), WO 97/35926 (Oct. 2, 1997); Farbstoffmischungen, enthaltend Thienyl- und/oder Thiazolazofarbstoffe; Dye mixtures containing thiophene and/or thiazole azo dyes Farbstoffmischungen, enthaltend Thienyl- und/oder Thiazolazofarbstoffe; Dye mixtures containing thiophene and/or thiazole azo dyes; C. Grund, H. Reichelt, A. J. Schmidt, F. Wxc3xcrthner, R. Sens, S. Beckmann, DE 19648564 A1 (May 28, 1998), DE 196 50 958 A1 (Jun. 10, 1998); WO 98/23688 (Jun. 4, 1998) Indoleninmethinfarbstoffe auf Basis von Trifluormethylpyridonen; Trifluormethylpyridone Based Indolenine Methine Dyes; Indoleninmethinfarbstoffie auf Basis von Trifluormethylpyridonen; Trifluormethylpyridone Based Indolenine Methine Dyes; F. Wxc3xcrthner, H.-W. Schmidt, F. Haubner, DE 19643097 A1 (Apr. 23, 1998), Vernetzbare Triarylaminverbindungen und deren Verwendung als Ladungstransportmaterialien; Crosslinkable triarylamines bearing ethynyl groups for use as charge transport materials; F. Wxc3xcrthner, R. Sens, G. Seybold, K.-H. Etzbach, DE 197 11 445 A1 (Sep. 24, 1998), WO 98/41583 (Sep. 24, 1998) Farbstoffsalze und ihre Anwendung beim Fxc3xa4rben von polymerem Material; Colorant Salts and their Use in Dying Polymeric Materials)
At website, www.sigma-aldrich.com and in their section immunochemicals of the catalogue xe2x80x9cBiochemicals and reagents for life sciences research, 2000-2001 of Sigma-Aldrich has described labeling reagents and cell linker labeling kits pp. 1454-1456. Three US patents by Horan, Paul K, Jensen, Bruce D, Siezak, Sue E, U.S. Pat. No. 4,783,401 of Nov. 8, 1988; Horan, Paul K, Siezak, Sue E in U.S. Pat. No. 4,762,701 of Aug. 9, 1988 and Horan, Paul K, Jensen, Bruce D, Siezak, Sue E in U.S. Pat. No. 4,859,584 dated Aug. 22, 1989 disclosed respectively xe2x80x9cviable cell labelingxe2x80x9d, in vivo cellular tracking and cell growth rate determination by measurement of changes in cyanine dye levels in plasma membranes.
Nontoxic and cell permeant fluorescent dyes are greatly in demand for their applications in the study of live cell functions, drug delivery, study of various cell organelle and many more. An eukaryotic cell may have several compartments each bounded by a membrane whereas a bacterial cell may consist of a single compartment. The permeability specificity is a characteristic of the cell membranes. A good dye is the one which can show the different parts of the cell at one emission and at different emissions (The Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the United States of America, 1996).
Dyes with fluorophore attached to various proteins have been synthesized and described in Handbook of fluorescent probes and Research chemicals by Richard P. Haughland 1996. Page 126-128. The range from the blue fluorescent Cascade blue and new AMCA-S dyes to the red fluorescent Texas Red dyes and phycobiliproteins is embodied. The company has high lighted Oregon green conjugates, Rhodol Green conjugates; Bodipy conjugates;Eosin labelled secondary reagents; Red fluorescent Rhodamine Rd-X and Texas Red-X conjugates; Phycobiliprotein conjugates; Cascade blue and AMCA-S conjugates.
All these dyes are synthetic and single one of them emits in one particular spectral range. The combinations of 2-3 dyes are then made for multiple colored experiments. The same company also offers Jasplakinolide, a macro-cyclic peptide isolated from the marine sponge Jaspis jolnstoni as a cell permeant F-actin probe. But it is toxic and exhibits fungicidal, insecticidal, and antiproliferative activity. Most of the currently available dyes in the market are synthetic. Stainfile-Dyes A has given a Dye index of 264 dyes. Out of which 258 are synthetic and only six are natural dyes. (http://members.pgonline.com/xcx9cbryand/dyes/dyes.htm).
Production of synthetic dyes often require use of strong acids, alkalis and heavy metals as catalysts at high temperatures. This makes the processes and the effluents to be discharged an issue of environment degradation. The dyestuff industry is continuously looking for cheaper and more environmentally friendly routes to existing dyes. (Hobson and Wales, 1998. Green Dyes, Journal of the Society of Dyers and colorists (JSDC), 1998,114,42-44).
All of the available dyes are not fluorescent. Bitplane products have displayed list of the 123 fluorochromes in the market and their excitation and emission spectrum (http://www.bitplane.ch/public/support/standard/fluorochrome.htm).
Fluorescence is a phenomenon in which an atom or molecule emits radiation in the course of its transition from a higher to a lower electronic state. It follows Stoke""s law, according to which the wavelength of the fluorescent radiation is always longer than that of the excitation radiation. The process of fluorescence is quite different from the phosphorescence and bioluminescence. The term fluorescence is used when the interval between the act of excitation and emission of radiation is very small (10xe2x88x928 to 10xe2x88x923 second). In phosphorescence, the time interval between absorption and emission may vary from 10xe2x88x923 second to several hours (R. Norman Jones, 1966 in: The encyclopaedia of chemistry, 2nd edition, 1966, Pages 435-436). Bioluminescence is the term used for the light produced as a result of a chemical reaction, occurred at a particular time in a particular cell within the body of a living organism.
A large number of fluorescent dyes are reported in xe2x80x9cThe Handbook of Fluorescent probes and Research Chemicalsxe2x80x9d by Richard P. Haughland, 6th edition Printed in the United States of America, 1996. In the same book on pages 1-6, Ian D. Johnson (1996) described in details the process of fluorescence and its methods of detection in certain molecules called iluorophores or fluorescent dyes by him (generally polyaromatic hydrocarbons or heterocycles). The most versatile currently in use fluorescent dyes are Fluorescein, fluorescein based, BODIPY dyes, and their derivatives. The authors have dealt in with the shortcomings of all these dyes and described their preferences of characteristics of dyes. Many derivatives of the fluorescent dyes and their synthesis are disclosed in US patents (Haughland, R. P and Kang, H. C. U.S. Pat. No. 4,774,339, published on Sep. 27, 1988; Haughland, R. P and Kang, H. C. U.S. Pat. No. 5,248,782 of Sep. 28, 1993; Kang, H. C. and Haughland, R. P, U.S. Pat. No. 5,187,288 published on Feb. 16, 1993; Kang, H. C. and Haughland, R. P in U.S. Pat. No. 5,274,113 of Dec. 28, 1993 and; Kang, H. C. and Haughland, R. P, U.S. Pat. No. 5,433,896 Jul. 18, 1995; Kang, H. C. and Haughland, R. P. U.S. Pat. No. 5,451,663 published on Sep. 19, 1995). Rosenblum Barnett B, Spurgeon S, Lee Linda G, Benson Scott C and Graham Ronald J in international patent No. W00058406, publication date Oct. 5, 2000 reported 4,7-Dichlororhodamine dyes useful as molecular probes.
R. Norman Jones in The encycopaedia of chemistry, 2nd edition, 1966, Pages 435-436 has described specialty of a good fluorophore. According to him A fluorescent molecule must have a good chromophoric system for absorption of excitation energy and a shielding mechanism to save too rapid dissipation of the excitation energy into vibrational motion before the fluorescence retardation act can occur. He also commented that though the relationship of the molecular structure and the fluorescence of compounds are not well understood there are certain groups, presence of which is associated with fluorescence. For example, in the organic molecule presence of phthalein and aromatic structures such as anthracene and naphthacene are particularly associated with bright fluorescence. Few inorganic compounds fluoresce strongly in the liquid state and in solids, fluorescence is often modified by the presence of trace impurities.
Pigments belong to the categories of inorganic and organic types. The formers are the inorganic chemistry compounds, which are used for various decorative and painting purposes etc. Organic pigments like organic dyes date back to the ancient times. The use of dyes from plants like Eirazil wood, log-wood, Persian berry indigo and madder are reported from near east and far eastern countries even before Biblical times (George L. Clark, 1966 xe2x80x9cEncyclopaedia of chemistry, 2nd ed. Pages 833-835).
Debra K. Hobsonand David S. Wales describe xe2x80x9cGreen dyesxe2x80x9d which are produced as secondary metabolites from some groups of living organisms like fungi, blue green algae, sea urchins, star fishes, arthropods and coral reef coelenterates (Journal of the Society of Dyers and Colourists (JSDC), 114, 42-44, 1998). These are anthraquinone compounds, historically of crucial importance in the dyestuffs industry.
Variety of carotenoid pigments are reported from the carotenoid-producing bacterial species (U.S. Pat. No. 5,935,808 published on Aug. 10, 1999, inventors Hirschberg, et al and U.S. Pat. No. 5,858,761, Jan. 12, 1999 inventors Tsubokura, et al). Collin; Peter Donald in his U.S. Pat. No. 6,055,936 published on May 2, 2000 disclosed sea cucumber carotenoid lipid fractions and process. Bandaranayake, W. M. and Des Rocher, A 1999 (Marine biology 133;163-169) described carotenoid pigments from the body wall, ovaries and viscera of Holothuiria atra from Australia.
All these colorants and dyes are however not fluorescent. Fluorescent dyes most of which are synthetics are disclosed in several international patents and US patents. These have been used in variety of applications. The amount of patents in this field shows the importance of these dyes. Synthetic parazoanthoxanthin A (m.w.214.2), emitting fluorescence at lambda (em) 420 nm, was found to be a pure competitive inhibitor of cholinesterases. Sepcic K, Turk T, Macek P (Toxicon, 36 (6): 937-940,1998). Welch; David Emanuel (U.S. Pat. No. 5,989,135 released on date Nov. 23, 1999 disclosed a luminescent golf ball.
White et al. (U.S. Pat. No. 6,110,566, dated Aug. 29, 2000 and WO9920688) described a flexible polyvinyl chloride film that exhibits durable fluorescent colors.
Dipietro Thomas C (International patent WO9938916) disclosed the use of fluorescent polymeric pigments in variety of paints, inks and textiles. Cramer Randall J (Patent No. EP0206718 published on Dec. 30, 1986) described a composition with fluorescent dye for bleaching and brightening of polymer.
Leak detection is another utility disclosed by some (Leighley; Kenneth C. U.S. Pat. No. 6,056,162 dated May 2, 2000). Cooper et al. U.S. Pat. No. 6,165,384 published on Dec. 26, 2000 disclose a full spectrum fluorescent dye composition for the same purposes. Lichtwardt et al. U.S. Pat. No. 5,902,749 dated May 11, 1999 use fluorescent dye in an automated chemical metering system.
There are reports on few fluorescent natural dyes available. A green fluorescent protein GFP has been described from the pacific jellyfish, Aequora aequora by Shimomura, O, Johnson, F. H. and Saiga, Y; in Journal of cellular and comparative physiology, 59, 223-239, 1962, Chalfie M in 1: Photochem Photobiol October 1995 62 (4): 651-6 xe2x80x9cGreen fluorescent proteinxe2x80x9d; Youvan D C, Michel-Beyerie ME xe2x80x9cStructure and fluorescence mechanism of GFP in National Biotechnology Oct. 14, 1996 (10): 1219-20 and Chalfie M, Yuan Tu, Ghia Euskirchen, William W. Ward, Douglas C Prasher in SCIENCE 263 (1994) 802-805 reported that GFP purified is a protein of 238 amino acids. It absorbs blue light maximally at 395 nm with a minor peak at 470 nm and emits green light at the peak emission of 509 nm with a shoulder at 540 nm. This fluorescence is very stable and virtually no photo bleaching is observed.
GFP with fluorescence in other wavelengths in the ranges of red and yellow are described from non bioluminescent anthozoans, particularly Discosoma coral. Gurskaya N G, Fradkov A F, Terskikh A, Matz M V, Labas Y A, Martynov V I, Yanushevich Y G, Lukyanov K A, Lukyanov S A in 1: FEBS Lett Oct. 19, 2001; 507 (1): 16-20n described GFP-like chromoproteins as a source of far-red fluorescent proteins. Wachter R M, Elsliger M A, Kallio K, Hanson G T, Remington S J. In 1: Structure Oct. 15, 1998; 6(10): 1267-77 described xe2x80x9cStructural basis of spectral shifts in the yellow-emission variants of green fluorescent proteinxe2x80x9d. Fradkov A F, Chen Y, Ding L, Barsova E V, Matz M V,Lukyanov S A xe2x80x9cNovel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence. In 1:FEBS Lett 2000 Aug. 18: 479 (3): 127-30. They describe a novel gene for advanced red-shifted protein with an emission maximum at 593 nm was cloned from Discosoma coral. The protein, named dsFP593, is highly homologous to the recently described GFP-like protein drFP583 with an emission maximum at 583 nm. They developed various mutants of both these genes. A hybrid mutant variant resulted I a mutant variant with a uniquely re-shifted emission maximum at 616 nm. Matz M V, Fradkov A F, Labas Y A, Savitsky A P, Zaraisky A G, Markelov M L, Lukyanov S A. 1: Nat Biotechnolo 1999 Dec: 17(10): 969-73.
In the south east and south pacific countries sea cucumbers are well known for their use in the health food and drug industry as a food item or ingredient to various drug compositions especially for inflammation of joints, sprains and other therapeutics. Several US and International patents are on record and screened. (Fan Hui-Zeng, Yu Song, Yamanaka E, Numata K, Oka T, Suzuki N, muranaka Y in U.S. Pat. No. 5,519,010 dated May 21, 1996; Weiman, Bernard, U.S. Pat. No. 5,888,514 published on Mar. 30, 1999; Katsukura, Kitazato, Kenji Yamazaki, Yasundo U.S. Pat. No. 5,993,797 dated Nov. 30, 1999; Henderson, R. W; Henderson, T and Hammd, T U.S. Pat. No. 6,255,295 dated Jul. 3, 2001; Shinya U.S. Pat. No. 6,203,827 of Mar. 20, 2001; U.S. Pat. No. 5,770,205 patent by Collin Peter Donald published on Jun. 23, 1998 and WO patent no. 0001399 published on Jan. 13, 2000; Kovalev V G, Sementsov V K, Slutskaja, Akulin V N, Timchishina G N in RU 2147239 published on Apr. 10, 2000; Li Zhaoming, Zhu Beiwei CN 1286926 dated Mar. 14, 2001 Qu Jianhong, Song Xiuqin, Zheng Fuqiang CN 1223131 dated Jul. 21, 1999; Wufa Zhuang Wufa, Meizheng Zhuang CN 1142365 dated Feb. 12, 1997; Fang Hua CN1312031 dated Sep. 12, 2001 and Ding Cunyi CN1173290 dated Feb. 10, 1998, Collin Peter Donald W09937314 published on Jul. 29, 1999). The use of the ingredients originated from sea-cucumber in Anti HIV drug are disclosed (Hoshino Hiroo EP 410002 dated Jan. 30, 1991 and Hoshino Hiroo EP495116 dated Jul. 22, 1992). In view of their importance the animals are tried to be cultivated under captivity (Annie Mercier, S C Battaglene and Jean-Francois Hamel in Journal of experimental Marine Biology and Ecology Volume 249 issue 1: 89-110. 2000 xe2x80x9cSettlement preferences and early migration of the tropical sea cucumber Holothuria scabra). Gu Zaishi, Wang Shuhai, Zhou Wei disclosed xe2x80x9cEcological reproducing method for Stichopus japonicus xe2x80x9cin patent No. CN1179261 dated Apr. 22, 1998.
But as all these patents are not directly of relevance to the topic of the present patent so they have been not included as reference.
Goswami, Usha and Ganguly, Anutosh has filed a patent on a natural fluorescent dye from a marine invertebrate (CSIR, NF-140/2001; U.S. patent application Ser. No.: 09/820654 filed on Mar. 30, 2001). This pertains to the crude extract from Holothuria scabra, which has the fluorescent qualities at three different wavelengths when excited at different UV and visible ranges of the spectra of light. The invention also provides a process of the extraction, purification and characterization of this new dye, which is a partially purified natural dye from a sea cucumber. The utilities of the dye as a epifluorescent stain and non-radioactive fluorescent dye useful for labeling of molecular probes for in situ hybridization studies is described besides several other qualities of the dye as a drug. In this patent prior art, we have dealt in details about the pigments, synthetic dyes and natural dyes from terrestrial plants and microbes. (U.S. Pat. No. 4,452,822 published on Jun. 5, 1984, inventors Shrikhande, Anil J., U.S. Pat. No. 5,321,268 of Jun. 14, 1994 by Crosby David A and Ekstrom Philip A; U.S. Pat. No. 5,405,416 published on Apr. 11, 1995 authors Swinton; Robert J, U.S. Pat. No. 5,858,761 published on Jan. 12, 1999, inventors Tsubokura, et al. U.S. Pat. No. 5,902,749 of May 11, 1999 inventors Lichtwardt et al. U.S. Pat. No. 5,908,650 published on Jun. 1, 1999 inventors Lenoble, et al. U.S. Pat. No. 5,920,429 published on Jul. 6, 1999 Burns et al. U.S. Pat. No. 5,935,808 on Aug. 10, 1999 of Hirschberg, et al; U.S. Pat. No.5,989,135 of Nov. 23, 1999 inventors Welch; David Emanuel; U.S. Pat. No. 6,055,936 of May 2, 2000; Collin; Peter Donald; U.S. Pat. No. 6,056,162 May 2, 2000; Leighley; Kenneth C.; U.S. Pat. Nos. 6,103,006 Aug. 5, 2000 DiPietro; Thomas C.; 6,110,566 Aug. 29, 2000 White et al. 6,140,041 Oct. 31, 2000 LaClair; James J. 6,165,384 Dec. 26, 2000 Cooper et al.; U.S. Pat. No. 6,180,154 Jan. 30, 2001 Wrolstad et al. EP0206718 published on Dec. 30, 1986 inventors Cramer Randall J; IE901379 of Jan. 30 1991 Lee Linda G; Mize Patrick D; WO9010044 of Jul. 7, 1990. Swinton; Robert J; AU704112 published on Oct. 7, 1997 inventors Burns David M; Pavelka Lee A; DE19755642 of Jun. 24, 1999 of Weimer Thomas D R.; WO9938919, Sep. 28, 1999 Laclair James J; WO0058406 of Oct. 5, 2000 by Rosenblum Barnett B et al.; WO9938916 of Aug. 15, 2000 inventors DiPietro; Thomas C; WO9920688 of Aug. 29, 2000 inventors Pavelka Lee et al.; WO9920688 of Aug. 29, 2000 inventors
White et al. The multiple uses oiF fluorescent dyes in the molecular biology research, in industrial applications and in life saving devices etc. are also described. Collin, P. D in his U.S patents of Jun. 23, 1998, Mar. 2, 1999 and Nov. 16, 1999 respective U.S. Pat. Nos. as 5,770,205, 5,876,762 and 5,985,330 have described therapeutic properties of various body parts of sea cucumber.
In another patent application, inventors of the present application i.e. Goswami, Usha and Anutosh Ganguly (U.S. Provisional application No. 60/317,190 filed on Sep. 6, 2001) has described a novel organosilicon Sixe2x80x94Oxe2x80x94R type of compound and multiple fluorescent natural dye purified from the body wall extract of a marine invertebrate Holothuria scabira. The compound is a polysacchride fluorochrome having a phenolic fluorophore part and is connected to a silicon matrix around it through the sulphate bonds. This silcon part is an integral part of the core molecule and takes part in the metabolism of the animal. The compound is rich in sulfur. The invention also provides a process for the extraction, purification and characterization of the novel compound and the multiple fluorescent dye from a living marine organism, especially sea cucumber. Further, the compound can be an easily miscible ingredient in compositions of Dye industry, Cosmetic industry and pharmaceutical industries.
In this patent a large body of literature upon fluorescent dyes reported in the Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the United States of America, 1996 are incorporated. In US and international patents many derivatives of the fluorescent dyes and their synthesis are disclosed (Haughland, R. P and Kang, H. C. U.S. Pat. No. 4,774,339, published on Sep. 27, 1988; Haughland, R. P and Kang, H. C. U.S. Pat. No. 5,248,782 of Sep. 28, 1993; Kang, H. C. and Haughland, R. P, U.S. Pat. No. 5,187,288 published on Feb. 16, 1993; Kang, H. C. and Haughland, R. P in U.S. Pat. No. 5,274,113 of Dec. 28, 1993 and; Kang, H. C. and Haughland, R. P, U.S. Pat. No. 5,433,896 Jul. 18, 1995; Kang, H. C. and Haughland, R. P. U.S. Pat. No. 5,451,663 published on Sep. 19, 1995, Rosenblum Barnett B, Spurgeon S, Lee Linda G, Benson Scott C and Graham Ronald J, international patent No. W00058406, publication date Oct. 5th, 2000 reported 4,7-Dichlororhodarnine dyes useful as molecular probes).
All these references pertain to the present patent also.
The applicants have adopted a different approach from their earlier patents and those reported by other workers.
The dyes reported in our earlier patents were though natural but they were toxic. The spectral ranges of emissions at various excitation wavelengths were also different. The dyes were toxic, and not suitable for in situ studies of live cells. Their applications and uses in the biomedical and engineering sciences were different.
The dye now reported is though also a natural dye but is a non-polar multiple fluorescent flurophore extracted from the female gonads of a non-bioluminescent invertebrate. The marine animal source is a holothurian, sea cucumber called Holothuria scabra which is a new source for a non-polar fluorescent dye. The dye is environmentally friendly, as unlike the synthetic dyes there is no need of intervening steps of strong acids and alkalis in their productions. Unlike the earlier described carotenoid pigments from the sea cucumber ovaries this dye is not a carotenoid pigment. It is a non-polar fluorophore easily separable from the conjugant biomolecule, which is a negatively charged protein.
Unlike most synthetic fluorescent dyes, our dye does not need to be mixed with another dye for getting different fluorescence hues at different wavelengths. They themselves emit six different colored fluorescence at three different excitation wavelengths, which can have multiple uses. The cell constituents show a contrasting staining from the background where only dye solution is present.
The present dye is cell permanent and permeates through plasma membrane, cytosol, nuclear membrane, nucleoplasm and chromosomes.
Once the dye attaches to the cell membranes it is stable at the room temperature for months and does not get photobleached and contaminated by microbes. Its fluorescence does not get deteriorated at high and low temperatures unlike extracts of some algae and luminescent organisms.
The dye is nontoxic to the E.coli bacteria and the sex cells and larvae of estuarine and marine animals. So its effluents will not kill the marine and estuarine animals larval stages. It is a nontoxic and eco-friendly dye.
Another important feature of the dye is that it shows fluorescence in only the live and fixed tissues. The dead ones are not stained and do not give any fluorescence. One important aspect of the dye is its making compositions and kits for non-radioactive in situ labeling of molecular probes and counter staining. At different wavelength excitations it gives the effect equivalent to color of DAPI, FITC and PI and other marketed fluorescent probes. The dye is a natural multiple fluorescent dye. Actually, this single dye covers the colors of wavelength spectrum of 123 flurochromes presently known in the market (see Bitplane products (Fluorochrome) on the Internet (http://www.bitplane.ch/public/support/standard/Fluorochrome.htm).
Yet another aspect is its use as a nontoxic fluorochrome stain in epiflourescence microscopy for the live cells. The dye is a natural dye and not synthetic which is permeant through various membranes and stains them differentially. This application provides a simple and quick method of checking cytogenetical preparations for multiple uses like molecular diagnostics using fluorescent in situ hybridization techniques, rapid diagnosis of bio-contamination in tissue cultures, food industry and industrial preparations, flow-cytometry etc.
Yet another aspect of the dye is its use as a component of the non-radioactive labeling kits for advanced molecular biology applications where protein dyes are needed for studies of live cell functions.
The present invention discloses a process of extraction, purification and characterization of a fluorescent dye solution from 70% alcoholic solution of ovarian tissue of a marine echinoderm. This is al natural dye, which has non-polar coloring part. Bioactive extract containing a natural non-polar and non-proteinaceous fluorescent dye extracted from alcoholic extract of the ovarian cells and a process of extraction, purification and characterization of a fluorescent pigment from ovarian extract of a marine echinoderm. The pigment is a natural dye, which is a non-polar fluorophore. The dye emits fluorescence in multiple fluorescent excitation ranges of UV and visible light spectra. It further discloses the chemical, physical, epifluorescent microscopic nature of the dye. The nontoxic, cell membrane permeable qualities are seen.
The main object of the present invention is to provide a natural nontoxic ecofriendly non-polar multiple colored fluorescent dye from the ovarian tissue of sea-cucumber Holothuria scabra. 
Another object of the invention is to provide a process for extraction, partial purification and characterization of the said dye from the marine animal Holothuria scabra which is a non bioluminescent marine invertebrate.
Another object of the invention is to provide a dye which is cell membranes permeable, giving demarcation of cell partitioning and having a visual effect at single and multiple emission ranges of UV and visible light spectral wavelengths.
Another object of the invention is to develop a dye with high quantum of fluorescence
Another object of the invention is to develop a dye with less photobleaching.
Another object of the invention is to develop a longer time photo stable dye at the room temperature.
Another object of the invention is to use the dye for checking survival and growth of animal and bacterial cells.
Yet another object of the invention is to provide compositions employing the dye obtained from the tissues of Holothuria scabra. 
Still another object of the invention is to provide a dye that emits fluorescence in six different wavelength ranges of UV and visible light spectra on particular excitation with three wavelengths.
Another object of invention is to observe the fluorescence and visible spectroscopic analysis and range of emission wavelengths.
Yet another object is to observe the six different fluorescence colored emissions of the dye in UV and visible ranges of epifluorescence microscopy cubes.
Still another object of the invention is to observe the effect of fluorescence staining of the cytogenetical slides to screen chromosomes, cells and tissues by using the dye of the invention.
Yet another object of invention is to see its nontoxic nature by performing experiments with bacteria.
Yet another object of invention is to see its nontoxic nature by performing experiments with marine animal cells.
Yet another object of the invention is its application for checking bacterial contaminations in food industry.
Yet another object of the invention is to observe the fluorescence and visible spectroscopic analysis and range of emission wavelengths.
Yet another object of the invention is that the dye is useful as a non-radioactive label of fluorescent molecular probes.
Still another object of the invention is that the dye does not get quenched fast in the excited light and photobleaching does not occur while screening slides.
Yet another object of the invention is to observe the fluorescence and visible spectroscopic analysis and range of emission wavelengths.
Yet, another object of the invention is that the dye is useful as a non-radioactive label of fluorescent molecular probes.
Another object of the invention is that the dye is highly stable at the room temperature of its own as well as when attached to the cell membranes.
Still another object of the invention is that its fluorescence quality does not get deteriorated even at extremely high and low temperatures.
Yet, another object of the invention is to develop kits for non-radioactive labeling of molecular probes and counter staining.
Yet, another object of the invention is industrial use of the fluorophore for synthesizing biomolecule-conjugants for flow-cytometry, micro-arrays, immunoassays and several other molecular applications as per the requirements of the researcher.
Still another object is to develop kits containing the fluorescent dye as in situ hybridization non-radioactive labelling kits for molecular probes.
Still another object is to develop low cost LIVE/DEAD Viability/Cytotoxicity kit for animal cells.
Still another object is to develop low cost LIVE/DEAD Viability/Cytotoxicity kit for bacteria.
Still another object is to develop low cost LIVE/DEAD Viability kit for sperms.
Still another object is to develop low cost LIVE/DEAD Viability kit for yeast cells.
Still another object is to develop low cost LIVE/DEAD Viability kit for animal cells.
Still another object is to develop low cost bacterial gram stain and LIVE/DEAD Viability kits.
Still another object is to develop low cost LIVE bacterial gram stain kit.
Still another object is to develop low cost bacteria counting kit.
Still another object is to develop low cost kits for animal cells with simple steps.
Still another object is to develop kits containing the fluorescent dye for cell culture contamination detection.
Still another aspect of the invention is the specificity and reliability. The live cells color and give fluorescence and the dead cells do not show any fluorescence.
Still another aspect of the invention is to use a single dye in the kits in place of presently used 2 dyes for live/dead cells detection.
Still another aspect of the invention is to develop less expensive and non-hazardous proliferation assays than the radioisotopic techniques.
Accordingly, the invention provides a bioactive extract containing a natural non-polar and non-proteinaceous fluorescent dye extracted from alcoholic extract obtained from ovarian cells of a non bioluminescent marine organism called sea-cucumber Holothuria scabra. The invention also provides a process for extraction, isolation and characterization of the said dye, which is a fluorophore detached from a protein conjugant. The said dye is partially purified and is in a solution form. The dye is tested for its nontoxic nature upon animal and bacterial live cells. The dye compositions will be useful as fluorescent epifluorescence microscopic stain, as fluorescent dye ingredient in multiple type of applications in the biomedical, molecular biology, microbiology, cell biology, recombinant technologies etc. and in formulation of new biomolecule conjugarits for applications in fluorescent probes.
After much research, the applicants have now identified a novel natural nontoxic fluorescent dye obtained from tissue of marine animals, especially from invertebrates and more specifically from the sea-cucumber Holothuria scabra.
The invention further provides a natural non-polar multiple fluorescent dyes which is obtained from the ovary of the animal and which is nontoxic to live cells. It also describes the physical and chemical nature of the dye and its stability in direct light, high and low temperatures. The said dye has six colored fluorescent emissions at three different excitation wavelengths of UV and visible light spectrum. The invention also relates to screening of cells under fluorescence microscope for a rapid check of contaminations and cell survival. The invention is also concerned with the uses of the dye as a non-radioactive label of protein, DNA and RNA molecular probes for advanced molecular diagnostics, epi-fluorescence microscopy for single double and multiple staining of chromosomes, cells and tissues, fluorescence in situ hybridization applications, and biocontamination check, in aquaculture and biomedical sciences for enhancement of fertility, as a component of kits where studies on live cells are required, novel remote sensing devices, underwater probes, life saving devices, mark the location of crashed aircraft, life rafts and Defence equipment for example rockets, various fluorescence applications in sub zero temperature conditions and many more. The dye is environmentally ecofriendly as it does not kill larvae of the estuarine and marine animals.
The invention describes fluorescent dye obtained from marine animals, which either absorb sunlight for their physiological functions or are exposed to longer duration of sunlight and appear to have evolved mechanisms of fluorescence at different wavelengths. Like the phytoplankton, picoplankton and photosynthetic bacteria absorb sunlight for their photosynthetic functions, the required wavelengths of light spectra are used in the chemical pathways and extra light is emitted following Stoke""s law.
The invertebrate animals who do not have an extra outer armor like a shell and conspicuous defence organs, who have hard and spiny skin, who have a strong endoskeleton formed of ossicles, are sedentary or slow mobility, have long hours of exposures to direct sunlight, live in sand or crevices may show fluorescence.
The present invention seeks to overcome the drawbacks inherent in the prior art by providing highly efficient and selective methods for extraction, purification and characterization of a dye from a marine invertebrate and its multiple uses in making kits for molecular diagnostics using non-radioactive labels, molecular markers, epiflourescence microscopy, component of new instrumentation devices for land and underwater probes, cosmetic industry, food industries and defense purposes etc.,
The said marine invertebrate is an echinoderm taxonomically called Holothuria scabra belonging to the class Holothuroidea. The product of the invention is a novel nontoxic multicolor fluorescent protein dye, which is reported for the first time. The animals were collected from the shores of central West Coast of India during low tide, brought to the laboratory and maintained in glass tanks containing seawater of salinity 30-32% per par. The animals were adults and sexually mature. The taxonomic position was identified as above.
In fact, most of the dyes available are synthetic in nature. There are only 6 types of natural dyes. This includes dyes obtained from all living organisms. The fluorescent dye reported in the present invention is the only one of its specialty of multicolor fluorescence nontoxic, living cell membrane permeant extracted from ovarian tissue of a marine sea cucumber and has abilities to be conjugated to biomolecules.
As used herein the term dye is used for a dye solution, which does not get decolorized by a reducing agent. The said dye imparts color to the fibre, cellulose, cell membranes etc. It is called a natural dye as the source is from a marine animal found commonly in the nature along shores, shallow and deep waters of the world and is not a synthetic pigment. A fluorescent dye is one, which on excitation at a particular wavelength during the transition from a higher to the lower electronic state within a very short duration it emits light.
Multiple colored fluorescence means the emission of different colored light when excited at different ranges of wavelengths. It emits blue, yellowish green and orangish red colored hues of fluorescence at excitations with different spectra of UV and visible light.
Cell membrane permeant means that the dye passes through the pores of the live cell and nuclear membranes of the cell and imparts it multicolored fluorescence in the shades of blue, bluish white, green, yellow orange and orangish red depending upon excitation with the UV, blue and green wavelength ranges.
The dye does not stain the already dead cells of both animal and bacteria.
The nontoxic to live cells means when tested upon live cells of both marine animal and bacteria (E.coli), their cells do not die.
Photostability at room temperature means when left on the bench at room temperature (xcx9c28 degree centigrade) the florescence quality does not deteriorate. Photostabilty of dye after attaching to cell membranes means the continuation of fluorescence emission after staining the live and fixed cells with the said dye.
The molecular diagnostics as used herein means the use of the dye as a non-radioactive label of molecular probes for fluorescent in situ hybridization applications in molecular cytogenetics and as markers in microarrays, and molecular biological studies.
The epifluorescent microscopy here pertains to the microscopic studies of cyto-genetical preparations of slides by using the present dye as a stain and recording different colored fluorescence when observed under different cube configurations emits a particular colored emission on excitation with known fluorochromes. The fluoro-chrome cubes WUB, WB, WG are the designated filter cube configurations of the Olympus BX-FLA reflected light fluorescence attachment for different wavelengths.
Accordingly, the present invention provides a bioactive extract containing non-polar and non-proteinaceous fluorescent dye obtained from alcoholic extract ovarian cells of a non bioluminescent marine organism Holothuria scabra occurring in intertidal, submerged, shallow and deep waters, usually abundant in shaded areas such as acloves, crevices, ledges, overhangings, rocky and sandy habitats; dull to bright coloured with or without exo- and endo skeleton, sessile, sedentary drifters, ektonic with varied swimming power usually nocturnal in habit, liabile to active predation, with and without luminescent and fluorescent pigments giving emissions in few to all wavelength ranges of UVB,UVA visible colored spectrums and infra red spectrum.
One embodiment of the invention relates, to the bioactive extract obtained from the marine organism, which is useful as a natural fluorescent dye having the following characteristics:
i. no decolorization by a reducing agent,
ii. it is a natural compound,
iii. crude extract of the dye is orange in color,
iv. semi purified dye solution is bright orange in color for the naked eye,
v. under tube light it emits variety of colors of the visible light spectrum,
vi. the pigment separated form protein part is insoluble in water and alcohol,
vii. the partially purified dye pigment is soluble in ether.
viii. the dye is a non-polar dye having pH 7.0,
ix. absence of reduciable chromophore,
x. dye solution emits six different colored fluorescence at 3 different wavelengths of the UV and visible ranges of the fluorescent cubes of an epifluorescence microscope, depending upon whether it is the dye solution alone or the cells on which dye has permeated and attached,
xi. fluorescence of blue color emission occur in the 450 nm-470 nm range on excitation under ultra violet cube WU-330 nm-385 nm excitation range,
xii. fluorescence of yellowish green color emission occurs in the 510 nm-570 nm range on excitation under WB cube of 450 nm-480 nm excitation range,
xiii. fluorescence of orange color emission occurs in the 610 nm-650 nm range on excitation under WG cube of 510 nm-550 nm excitation range,
xiv. the dye emits hues of yellowish grays under the ordinary transmitted light bulb of the epifluorescence microscope when seen under 100xc3x97oil immersion objective,
xv. the dye emits fluorescence colors even at a dilution range of 1:40000 times
xvi. the fluorescence of the extract persisted even up to 1 year when preserved at about 4xc2x0 C.,
xvii. the fluorescence of the dye is highly photostable and does not get deteriorated by long exposures to direct light,
xviii. the fluorescence of the dye does not change even when frozen at xe2x88x9220xc2x0 C., a temperature at which the molecules are unable to attain the energy necessary for activation like in extracts from luminescent organisms,
xix. the dye is nontoxic to the living cells of eukaryotes and prokaryotes (E. coli)
xx. the dye is permeable to cell membranes,
xxi. the dye is impermeable to dead eukaryotic cells and dead prokaryotic cells, and
xxii. the dye is non degradable even after staining the cell components.
Another embodiment of the invention, the multicolored emissions of the dye at different wavelengths of excitations are comparable to the fluorochrome microscopic stains available in the market.
Still another embodiment, the blue colored fluorescence of the present dye is comparable to the emission of same color by DAPI fluorochrome at the same wavelength excitation, used as components of the non-radioactive labeling kits of biochemistry, cell biology, immuno-chemistry and molecular biology.
Still another embodiment, the yellow colored fluorescence of the dye under the visible range is comparable to the same colored emissions of Auramin used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
Yet another embodiment, the yellow colored fluorescence of the said dye in the visible range is comparable to the same colored emissions of FITC used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry and molecular biology.
Yet another embodiment of the invention provides a dye having orange colored fluorescent emission, comparable to the orange fluorescence color of Propidium Iodide fluorochrome which is used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
Yet another embodiment of the invention provides a dye having orange colored fluorescent emission, comparable to the orange fluorescence color of Rhodamine fluorochrome used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
Yet another embodiment of the invention provides a dye having orange colored fluorescent emission, comparable to the orange fluorescence color of TRITC fluorochrome used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
Yet another embodiment of the invention provides a stable dye at room temperature and has a long shelf life.
Yet another embodiment of the invention the molecular and radioactive kits of the said dye can be exported at the room temperatures.
Yet another embodiment of the invention provides a dye having characteristics of at least one hundred different fluorochromes available in the market namely DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine, Rhodamine, TRITC, and propidium iodide, etc.
Yet another embodiment of the invention provides a dye used in all applications in place of Phycobiliproteins.
Yet another embodiment of the invention, the said dye under bright field of fluorescent microscope when seen under 10xc3x97objective, the hues of bluish grays produce a phase contrast effect which is useful in rapid screening of cytogentical, cytological, and histochemical slides and save expenses on the extra phase contrast accessory component of microscope.
Yet another embodiment of the invention, the said dye under 100xc3x97oil immersion objective of an ordinary transmitted light microscope the proteins of yolk, nucleoplasm and chromatin of actively dividing cleavage cells show different colors of staining in the hues of brownish yellow for former, yellow for the latter and dark blue for the last cell component, which is useful in rapid bioassays of effect can be seen on the various histochemical components of the cells.
Yet another embodiment of the invention, the fluorescence color emissions follow Stoke""s law of fluorescence.
Yet another embodiment of the invention, the microphotographs with Kodak film rolls show hues of the adjacent color emission wavelengths such as blue color fluorescence under the epifluorescence.
Yet another embodiment of the invention, the microphotographs with Kodak film rolls shows hues of the adjacent color emission wavelengths like when seen yellow color fluorescence under the epifluorescence microscope in microphotograph the hues of green also found.
Yet another embodiment of the invention, orange fluorescence color is seen under the epifluorescence microscope in microphotograph, the hues of red also found.
Yet another embodiment, the cytogenetic slides seen under all fluorescences gives a counterstain effect of cells and cell components versus the background color where no specimen but only dye is present.
Yet another embodiment, the diluted dye with water in the ratio above 1:4,50,000 to 1:9,00,000 times give fluorescence of six colors at three different wavelengths.
One more embodiment of the invention provides compositions comprising bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with suitable additives for the following useful applications:
i. preparing fluorescent color coating compositions and inks useful in variety of paints, inks, textile;
ii. as a fluorescent molecular probe in situ hybridization kits for molecular diagnostics;
iii. used in experiments where various applications of fluorescent dyes are needed to be performed at field stations situated at subzero degree temperature areas;
iv. useful as fluorochrome stains for epifluorescence microscopy;
v. useful in cell permeant dye compositions;
vi. a composition of fluorescent dye for bleaching and brightening polymer;
vii. leak detection with a full spectrum fluorescent dye;
viii. use in automated chemical metering system;
ix. to mark location of crashed air-crafts, life crafts, and equipment for example rockets;
x. under sea probes;
xi. chromatophore sunscreen component of cosmetics creams and lotions;
xii. fluorescent in situ hybridization application kit component for molecular diagnostics;
xiii. component of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology for labeling of DNA, RNA, Proteins and enzymes;
xiv. immunofluorescent detections;
xv. counterstain of DIG-labeled oligonucleotide probes and Anti-DIG Fab-fragments;
xvi. single and multiple flow cytometry applications;
xvii. fluorochrome stains for epifluorescence microscopy;
xviii. for a quick check of biocontamination in the health food industry, cosmetic industry, pharmaceutical and chemical industries;
xix. for rapid estimations of biocontaminants in laboratory cultures;
xx. for a rapid check of biopollutants under field conditions;
xxi. for a rapid check of dead and live bacteria E.coli; 
xxii. a natural colorant;
xxiii. a bioactive composition of the fluorescent dye in the ratio of 1:400000 in ether is sufficient to produce fluorescences of six colors at three different wavelengths and a phase contrast effect under transmitted light;
xxiv. a dye for various fluorescent applications to be performed in areas of sub zero temperatures;
xxv. a dye for making conjugants with variety of biomolecules;
xxvi. for cell permeant membrane dye compositions;
xxvii. for identification of dead and live cells in tissue cultures;
xxviii. for dye compositions in biosensors;
xxix. as dye composition in molecular and microbiological kits; and
xxx. a non-polar dye for detection of lipids and oils.
One more embodiment of the invention relates to a process for preparation of a bioactive extract containing a natural fluorescent dye from Holothuria scabra sea cucumber, said process comprises the steps of:
a) collecting the marine organism from seashore,
b) maintaining the organism in tanks containing seawater without any mechanical aeration overnight,
c) dissecting the washed animals and removing the female gonads,
d) extracting with 70% ethyl alcohol to obtain yellowish orange solution,
e) repeating the extraction steps 3-4 times to obtain colored solution,
f) filtering the above solution through Whatman No. 1 filter paper to obtain partially purified extract,
g) dissolving the extract obtained in step (f) in solvent ether, and
h) filtering through Whatmaini No 1 filter paper to obtain clear yellow colored solution named as xe2x80x9cNon-polar fluorescent dye solution
Another embodiment of the invention, the said dye solution is diluted further with water/seawater, in the ratio ranging between 1:400,000 to 1:900,000 times give fluorescence of six colors at three different excitation wavelengths.
Still another embodiment of the invention, wherein bioassays are conducted by using dilutions of the bioactive extract in the range of 1:450000, 1:200000, 1:100000, 1:50000 and 1:25000 times for assessing non-toxic nature of the dye upon survival of eukaryotic and prokaryotic cells.
The present invention provides a method for extraction, partial purification and characterization of a natural non-polar nontoxic cell membrane permeant multiple fluorescent dye. It further provides compositions at which the dye can detect live eukaryotic and prokaryotic cells without killing them and it comprises of:
collection of the material from field and maintenance in the laboratory conditions,
extraction of the pigment from the 70% alcoholic extract of the ovary of the echinoderm sea cucumber
Holothuria scabra, and partial purification of the dye.
Testing of biological activities.
The bioactive extract of the invention is obtained from 70% alcoholic extract of the ovarian tissue of marine sea-cucumber Holothuria scabra. This extract is useful as a natural fluorescent dye and has the following characteristics:
1) no decolorization by a reducing agent,
2) not a synthetic compound,
3) crude extract of the dye is yellowish orange in color,
4) the dye has two steps of its extraction from the ovarian cells i.e. at step-1 the alcoholic solution is extracted and left to evaporate and step-2, solvent ether is added to the dried mass and filtered through Whatmann filter 1.
5) it produces dye solution containing partially purified fluorophore.
6) the dye solution is bright orange in color.
7) under tube light it emits variety of colors of the visible light spectrum.
8) the pigment is insoluble in water and alcohol.
9) the pure dye pigment is soluble in solvent ether, the further dilutions can be made by following the procedures given.
10) is a non-polar dye,
11) has pH of about 7,
12) absence of a reducable group,
13) the fluorophore is associated with the protein which is removed when heated and coagulated,
14) dye in solution emits six different colored fluorescence at 3 different wavelengths of the UV and visible ranges of the fluorescent cubes of an epifluorescence microscope, depending upon whether it is the dye solution alone or the cells on which it has attached.
15) fluorescence blue color emission occur in the 450 nm-470 nm range when excited under ultra violet cube WU-330 nm-385 nm excitation range,
16) fluorescence yellowish green color emission occurs in the 510 nm-570 nm range when excited under WB cube of 450 nm-480 nm excitation range,
17) fluorescence orange color emission occurs in the 610 nm-650 nm range when excited under WG cube of 510 nm-550 nm excitation range,
18) the dye emits hues of yellowish grays under the ordinary transmitted light bulb of the epifluorescence microscope when seen under 100xc3x97oil immersion objective,
19) the dye emitted these fluorescence colors even at a dilution range of 1:400000 to 1:900,000 times and above.
20) the fluorescence of the extract persisted even after at least 1 year at the 4 degree centigrade
21) the fluorescence of the dye is highly photostable and does not get deteriorated by long exposures to direct light once the cells are stained even at the room temperature.
22) the fluorescence of the dye does not change even when frozen at minus 20 degree centigrade, a temperature at which the molecules are unable to attain the energy necessary for activation like in extracts from luminescent organisms.
23) The dye is nontoxic to the living cells of eukaryotes.
24) The dye is also nontoxic to the prokaryotes (E.coli)
25) The dye is cell membrane penmeant
26) The dye is cell membrane impermeable to dead animal cells.
27) The dye is cell membrane impermeable to dead E.colibacteria.
28) The dye is a non-degradable stain of the cell membranes.
The physical and other characteristics of the dye may be assessed by the following step:
a. color and solubility of the dye,
b. photostability
c. photobleaching
d. nontoxicity
e. selective staining of cell membranes
f. Physical checking of emission under a UV transilluminator 260-280 nm range,
g. Preparation of slide with live oyster eggs and sperms
h. Preparation of slides of live bacteria
i. Preparation of the fixed cells slides by air dried method,
j. Staining of slides with the dye,
k. Keeping controls for each experiment without adding dye.
l. Epifluorescent microscopic screening of the live eukaryotic cells slides under fluorochrome cubes WU, WB, WG and Bright field,
m. Epifluorescent microscopic screening of the fixed eukaryotic cells under fluorochrome cubes WU, WB, WG and Bright field,
n. Epifluorescent microscopic screening of the dead eukaryotic cells under fluorochrome cubes WU, WB, WG and Bright field,
o. Epifluorescent microscopic screening of the live bacterial cells under fluorochrome cubes WU, WB, WG and Bright field,
p. Epifluorescent microscopic screening of the dead bacterial cells under fluorochrome cubes WU, WB, WG and Bright field,
q. Epifluorescent microscopic screening of the live control cells without the dye under fluorochrome cubes WU, WB, WG and Bright field,
r. Microphotography of emitted fluorescence in the areas of slides without any cells,
s. Microphotography of emitted fluorescence of the cytogenetic slides under fluorochrome cubes WU, WB, WG and Bright field, and
t. Checking of wavelength ranges of the fluorescent hues of emission and wavelength ranges of the excitation ranges of fluorochrome cubes with the dye
u. Checking of wavelength ranges of the fluorescent hues of emission and wavelength ranges of the excitation ranges of fluorochrome cubes with the cells stained with the dye
v. Checking for cell membrane permeability of plasma membrane, cytoplasm, nuclear membrane, nucleoplasm and chromosomes.
Thus the invention provides a non-polar natural fluorescent dye of marine animal origin which emits six different colored fluorescence in the hues of blue, yellow and orangish red when excited with three different ranges of wavelengths in the UV and visible light spectral cubes of an epifluorescence microscope. The range of emissions of the dye solution and that of cells stained with the dye differs. The invention further relates to the epifluorescence microscopy of eukaryotic and prokaryotic live, fixed and dead cell preparations by using this dye as the epifluorescent microscopic stain. This dye could be used in making non-radioactive labeling kits for molecular diagnostics by fluorescent in situ hybridization in various molecular, biomedical and engineering sciences.
In an embodiment the source of the dye is an invertebrate marine animal belonging to SubKingdom: Metazoa, Phylum Echinodermata; subphylum: Eleutherozoa, Class Holothuroidea. Name: Holothuria scabra 
In yet another embodiment the Holothuria scabra is selected from the group comprising of sea cucumbers and widely distributed in the shores, shallow waters, deep waters all over the world particularly Indo-Pacific. The nearest well-known relatives of sea cucumber are the sea urchins and starfishes etc.
In yet another embodiment Holothuria scabra is dissected, its ovaries are separated and weighed. In step 1, to 1 mg of ovarian tissue by wet weight 3 mlxc3x973 times of 70% alcohol is added. This is called 70% alcoholic extract of ovarian tissue.
In yet another embodiment in step 2, heating the solution coagulates the protein and alcohol is evaporated. The dried mass dissolves only in non-polar solvent ether.
In yet another embodiment, the colored solution is partially purified by filtering through Whattman No. 1 filter paper.
In yet another embodiment the vial with screw cap carrying the solution of the dye is labeled as xe2x80x9cnon-polar dye solutionxe2x80x9d and stored in cold room at 4xc2x0 centigrade.
In yet another embodiment, the color of the dye solution is noted with naked eyes and tube light.
In yet another embodiment, the dye is found to be soluble in ether. The further dilutions for experiments can be made following given procedures.
In yet another embodiment, the photostability of the dye at the room temperature in the solution and on the stained cells is noted. It showed that the dye is non-degradable both at the room temperature and also once it attaches to the cell membranes.
In yet another embodiment test for electric charge of the Dye by electrophoresis is performed, the yellow spot showed non-polar behaviour and does not move towards any pole.
In yet another embodiment, the pigment is a dye as it is giving a color to the filter paper and staining the cell membranes.
In yet another embodiment, the dye has a pH of 7.0.
In yet another embodiment 1 ml of the dye solution of 70% alcoholic extract from which the present dye is isolated was taken and 0.2 gms of di thio erythritol was incorporated. The solution does not get decolorized. It showed that the reducible group is absent in the coloring part of the solution.
In yet another embodiment 9 ml of the 70% alcoholic extract from which the present dye is isolated was subjected to heating in a water bath at 100-degree Centigrade. Coagulation was observed. Which confirmed the presence of protein in the step 1 solution of the dye.
In yet another embodiment, after removal of the protein from the starting material, the colored part gets separated and dissolves only in ether.
In yet another embodiment, the alcoholic extract was subjected to Anthrone test (Ref) by using 4 ml of sulfuric acid and Anthrone reagent. 1 ml of Water 4 ml of conc. sulfuric acid and Anthrone reagent was kept as a blank. The blank was faintly green after 5 minutes whereas the extract was turned bright green after 5 minutes. The test proved that the carbohydrate is present in the alcoholic extract.
The fluorescence activity of the dye solution is due to a colored component that includes non-polar fluorophore attached to a negatively charged protein, which gets easily separated when the protein is coagulated.
In yet another embodiment nontoxicity test of the dye on eukaryotic cell survival was performed.
The dye was tested for cytotoxicity upon the oyster sperms. The survival of the sperms in the experimental set ups was taken as a parameter for showing nontoxicity. Male gonads of an oyster were removed and sperms were released in 100% seawater. These were filtered through muscline cloth to remove any debris. 1 mililiter (ml) of the sperm solution was Oaken and different concentrations (1 xcexcl, 2 xcexcl, 3 xcexcl, 4xcexcl and 5 xcexcl) of the Dye solutions were added. At every half an hour the observations of survival of the sperms were made under a microscope. The experiments were continued for 24 hours. The Controls were maintained without addition of Dye. It was seen that there was no effect upon the survival rates of sperms with addition of the dye. This proved nontoxic nature of the dye.
In yet another embodiment nontoxicity test of the dye was performed upon survival of prokaryotes. The extract was tested for cytotoxicity upon gram negative E.coli bacteria by observing their survival or mortality. A drop of the live E.coli bacteria in water (50 xcexcl) was placed on a microscopic slide. To this was added 0.5 xcexcl-1.0 xcexcl of the ether extract. The mixture was mixed using stirring needle. The slide was sealed so as to save it from evaporation. It was seen under the microscope that the bacteria remained alive for 24 hours till they remained in solution. They died if the solution got dried. The control experiments were performed. This proved that the dye is nontoxic to the bacteria.
In yet another embodiment, the bacteria do not show agglutination behavior. In another embodiment, the oyster eggs, sperms do not show agglutination behavior.
The applicants studied the nature of the dye and found that it gave multicolored emissions at different wavelengths of excitations, which are comparable to the fluorochrome microscopic stains already in the market. The blue colored fluorescence of the present dye is comparable to the emission of same color by DAPI fluorochrome at the same wavelength excitation, used as components of the non-radioactive labeling kits of biochemistry, cell biology, immunochemistry, and molecular biology. The yellow colored fluorescence of the said dye in the visible range is comparable to the same colored emissions of auramine used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
The yellow colored fluorescence of the said dye in the visible range is comparable to the same colored emissions of FITC used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
The orange colored fluorescent emission is comparable to the orange fluorescence color of Propidium Iodide fluorochrome used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
The orange colored fluorescent emission is comparable to the orange fluorescence color of TRITC fluorochrorrie used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
The dye is stable at the room temperature and has a long shelf life. The molecular and radioactive kits of the said dye can be exported at the room temperatures. The dye has characteristics of at least one hundred and twenty three different fluorochromes namely DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine, Rhodamine, TRITC, and propidium iodide etc., which are now in the market (Bitplane products). The dye, under ordinary light of microscope the hues of grays produce a phase contrast effect which is useful in rapid screening of cytogentical, cytological, and histochemical slides and save expenses on the extra phase contrast accessory component of microscope. The fluorescence color emissions follow Stoke""s law of fluorescence.
In yet another embodiment the epifluorescence microscopic studies are made by using this dye as a stain in the dilutions of above 1:400000 and recording emissions of light when excited by different cubes and compared the color hues with the known fluorochromes.
In yet another embodiment the screening was done using excitations of UV light and visible light spectra by WU, WB, WG and BF cubes of the Olympus reflected light.
In yet another embodiment WU cube""s wavelength range is 330 nm-385 nm.
In yet another embodiment WB cube""s wavelength range is 450 nm-480 nm.
In yet another embodiment WG cube""s wavelength range is 510 nm-550 nm.
In yet another embodiment BF is for the bright field where an ordinary tungsten bulb delivers light.
In yet another embodiment, the emission ranges of the dye at different excitation ranges were found out. The background of the eggs in the epifluorescence microscopy photos shows the emission color of the dye.
In yet another embodiment it was seen that excitation with the WU 330 nm-385 nm range emitted fluorescence in the 450 nm-470 nm range.
In yet another embodiment Excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 510-nm-570 nm range.
In yet another embodiment, the excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610-nm-650 nm range.
In yet another embodiment With BF the shades of yellowish grays were seen.
In yet another embodiment, the emission ranges of the dye after staining to the cell membranes at different excitation ranges were found out.
In yet another embodiment, the dye was used as fluorescence microscopic stain on the dead, live and fixed eggs of the oyster. The slides were screened under a epifluorescence microscope. It was noticed that the dead cells do not take up dye and shows no fluorescence.
In yet another embodiment, the dye was used as fluorescence microscopic stain on the live eggs of the oyster. The slides were screened under a epifluorescence microscope. It was noticed that the live cells showed fluorescence. The excitation spectral range and the emitted fluorescence strictly followed the Stoke""s law. These ranges were different from the emission ranges of the dye, which represents the background of the fluorescing cell.
In yet another embodiment the dye was used as fluorescence microscopic stain on the fixed eggs of the oyster in 3:1 ethanol and acetic acid fixative. The slides were screened under a epifluorescence microscope. It was noticed that the fixed cells showed fluorescence. The excitation spectral range and the emitted fluorescence strictly followed the Stoke""s law. These ranges were different from the emission ranges of the dye, which represents the background of the fluorescing cell.
In yet another embodiment excitation with the WU 330 nm-385 nm range emitted fluorescence in the 470nm-500 nm range in the cells.
In yet another embodiment excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 570 nm-610 nm range.
In yet another embodiment, the excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610 nm-650 nm range.
In yet another embodiment the epifluorescent microscopic screening of the dead eggs under Bright Field emitted light in full white range of the visible spectra and depending upon the density of the cell ingredients gave hues of yellowish grays like a phase contrast effect.
In yet another embodiment the epifluorescent microscopic screening of the live eggs under Bright Field emitted light in full white range of the visible spectra and depending upon the density of the cell ingredients gave hues of yellowish grays like a phase contrast effect.
In yet another embodiment the epifluorescent microscopic screening of the fixed eggs (3:1 ethanol:acetic acid fixative) under Bright Field emitted light in full white range of the visible spectra and depending upon the density of the cell ingredients gave hues of yellowish grays like a phase contrast effect.
In yet another embodiment, the dye was used as microscopical stain for the E.coli. One loop of live E.coli bacteria was placed in 50 microliter of water on a microscopic slide and mixed. To this was added 0.5-1 microlitre (1 xcexcl) of the dye solution towards outskirt of the bacterial suspension drop. The ether in the extract was allowed to evaporate by leaving the slide on bench for 4-5 sec. Both the drops were then mixed and a coverslip was placed on the sample and immediately sealed. Similarly a control preparation of bacterial suspension in water without dye was made.
In yet another embodiment both the slides were screened under oil immersion objective of a epifluorescence microscope (100xc3x97objective, 10xc3x97eye lens) for checking fluorescence. It was noticed that the dead cells don""t take up dye and shows no fluorescence.
In yet another embodiment the live bacterial cells showed fluorescence. The excitation spectral range and the emitted fluorescence wavelengths strictly followed the Stoke""s law. These were different from the dye solution and was as given below:
In yet another embodiment excitation with the WU 330-nm-385 nm range emitted fluorescence in the 470 nm-500 nm ranges.
In yet another embodiment Excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 570-nm-610 nm range.
In yet another embodiment, the excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610-nm-650 nm range.
In yet another embodiment, the control E.coli without any dye also showed no fluorescence.
In yet another embodiment microphotography of the slides with the dye used as epifluorescence microscopy stain was done.
In yet another embodiment The microphotography of emitted fluorescence in the areas of slides with cells and the surrounding background which represented the emissions of colors only due to dye was done, under WU 330 nm-385 nm range, with Kodak film of 400 ASA speed with an exposure time varying from 50 to 60 seconds.
In yet another embodiment the microphotography of emitted fluorescence in the areas of slides with cells and the surrounding background which represented the emissions of colors only due to dye was done, under WB 450 nm-480 nm range, range, with Kodak film of 400 ASA speed with an exposure time varying from 50 to 60 seconds.
In yet another embodiment the microphotography of emitted fluorescence in the areas of slides with cells and the surrounding background which represented the emissions of colors only due to dye was done, under WB 510 nm-550 nm range, with Kodak film of 400 ASA speed with an exposure time varying from 50 to 60 seconds.
In yet another embodiment the microphotography of emitted fluorescence in the areas of slides with cells and the surrounding background which represented the emissions of colors only due to dye was done, under Bright field with Kodak film of 400 ASA speed with an exposure time varying from 50 to 60 seconds.
In yet another embodiment permeation of dye in the Cell membranes was found out. The unfertilized, fertilized eggs and larvae of oysters were stained with the dye and egg suspension ratio of 1:50 microliter and screened under a fluorescent microscope. It was seen that the fluorescence was noticeable in the plasma membrane, nuclear envelope and chromatin. Though the wavelength ranges of emissions were the same and the colors were the hues of the same shade, there was a noticeable demarcation of boundaries of these parts of the cell. This proved that the dye is permeable through the live and fixed cell membranes of egg plasma membrane, cytoplasm, nuclear membrane, nucleoplasm and chromatin.
In yet another embodiment, the absence of fluorescence of these parts of the cell in the dead cells showed that dye is irnpermeant to dead cell membranes.
Different stains are used for different excitation cubes of the fluorescent microscope. For example DAPI (DNA staining, emits blue color), Fluorescein-dUTP; Hoechest 33258, 33342 are seen under excitation with 330 nm-385 nm excitation cubes; FITC, Acridine Orange (for DNA, RNA emits greenish/yellowish hues), Auramine under 450 nm-480 nm excitation cube and Rhodamine, TRITC and Propidium iodide (DNA, emits orange hues) under 510 nm-550 nm excitation cube.
In an embodiment to this epifluorescence putting a drop of the diluted extract and excitation with the WU filter having spectral range of 330-385 nm wavelengths does microscopic screening of the cytological slides.
In another embodiment epifluorescence microscopic screening of the cytological slides is done by putting a drop of the extract and excitation with the WB filter having spectral range of 450 nm-480 nm wavelengths.
In another embodiment epifluorescence microscopic screening of the cytological slides is done by putting a drop of the extract and excitation with the WG filter having spectral range of 510 nm-550 nm wavelengths.
In yet another embodiment epifluorescent microscopic screening of the cytological slides under Bright Field objective using this dye by transmitted light.
In yet another embodiment epifluorescence microscopic screening of the cytological slides stained with the dye is done by observing hues of the fluorescence color emitted by the respective excitations.
In another embodiment the excitation with the WU 330 nm-385 nm range emitted fluorescence is in the 470 nm-500 nm range.
In another embodiment, the excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 570 nm-610 nm range.
In yet another embodiment the excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610 nm-650 nm range.
In another embodiment epifluorescent microscopic screening of the cytological slides under Bright Field by using transmitted light emitted light in full white range of the visible spectra depending upon the density of the cell ingredients and giving a phase contrast effect.
In still another embodiment, the dye is diluted in 70% ethyl alcohol 1:9000 times and vaporized and the dried matter is again dissolved in 3 ml ether by repeating dissolution in the same amount of ether three times and further mixed in water to 1:50 time which means total dilution of the pigment is above 1:400000 times which gives fluorescence of six colors at three different wavelengths.
In yet another embodiment the invention provides a bioactive composition containing an extract obtained from the marine sea-cucumber Holothuria scabra in the ratio of 1:400000 to obtain fluorescence of six colors at three different wavelengths and a phase contrast effect under transmitted light.
In an embodiment, the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in the preparation of coating compositions and inks.
In another embodiment, the invention provides composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in detection of leaks.
In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in undersea probes.
In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful as a fluorescent probe in situ hybridization kits for molecular diagnosis.
In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful as a component of non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry and molecular biology.
In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in immuno fluorescent detections.
In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful as a counterstain of DIG-labeled oliogonucleotide probes and anti-DIG Fab-fragments.
In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in single and multiple cell quantitative fluorescence in flowcytometry.
In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful as fluorochrome stains for epifluorescence microscopy.
In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful for a quick check of biocontamination in the health food industry, cosmetic industry, pharmaceutical and chemical industries.
In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful for rapid estimations of biocontaminants in laboratory cultures.
In yet another embodiment the invention provides a composition comprising a bio active extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful for a rapid check of biopollutants under field conditions.
In yet another embodiment the invention provides a composition comprising a bio active extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in microbial kits.
In yet another embodiment the invention provides a composition comprising a bio active extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful as a natural colorant.
In yet another embodiment the dye solution contains a fluorophore which can be conjugated with proteins and other bio molecules for making customer oriented dye compositions.
TABLE-1 The Emissions of the different colored fluorescence of the fluorescent dye when excited with different wavelength fluorescent filter cubes of the Olympus epifluorescence microscope with dye solution and when attached to cell membranes of the prokaryotic cells.
TABLE-2 The Emissions of the different colored fluorescence of the fluorescent dye when excited with different wavelength fluorescent filter cubes of the Olympus epifluorescence microscope with dye solution and when attached to cell membranes of the eukaryotic cells.
Table-3 Table showing various commercially important features of the three natural fluorescent dyes extracted from the sea cucumber of the present invention.